1. Field of the Invention
This invention relates to the use of transforming growth factor-beta (TGF-.beta.) to induce bone growth in vivo and to formulations of TGF-.beta. and tricalcium phosphate useful for this purpose.
2. Description of Related Art
The disorders associated with bone loss present major public health problems for Western societies. Osteoporosis alone may affect 20 million Americans in the early years of the next century. Hence, there is wide interest in identifying factors or potential therapeutic agents that inhibit bone loss and stimulate the formation of healthy new bone.
Bone is an extremely complex, but highly organized, connective tissue that is continuously remodeled during the life of an adult by cellular events that initially break it down (osteoclastic resorption) and then rebuild it (osteoblastic formation). This remodeling process occurs in discrete packets throughout the skeleton, i.e., in both cortical bone and trabecular bone. It has recently been reported that mouse bone marrow cells can be stimulated to generate osteoclasts in the presence of parathyroid hormone-related protein or vitamin D. See Akatsu et al., Endocrinology, 125: 20-27 (1989); Takahashi et al., Endocrinology, 123: 2600-2602 (1988) and Takahashi et al., Endocrinology, 123: 1504-1510 (1988).
The currently available therapeutic agents known to stimulate bone formation are fluoride, estrogen, and vitamin D. Fluoride clearly increases trabecular bone mass, but questions remain about the quality of the new bone formed, the side effects observed in some patients, whether there are beneficial effects on vertebral fracture rates, and whether increased fragility of cortical bone with subsequent propensity to hip fracture follows.
Another approach is using agents that promote resorption (parathyroid hormone) and then interrupt resorption (calcitonin). One proposed, but not validated, such sequential therapeutic regimen is coherence therapy, where bone metabolic units are activated by oral phosphate administration and then resorption is inhibited by either diphosphonates or calcitonin.
Within the past few years several factors that stimulate osteoblasts were identified in bone, including TGF-.beta., fibroblast growth factor, platelet-derived growth factor, insulin-like growth factor I, and .beta.2 macroglobulin. Of these, TGF-.beta. and IGF-I were deemed attractive candidates for factors linking previous bone resorption with subsequent bone formation. Mundy, The Journal of NIH Research, 1: 65-68 (1989).
Other proteins stored in the bone matrix may also be important for bone formation. When demineralized bone was injected into the muscle or subcutaneous tissue of rats, a cascade of events, including chondrogenesis, ensued. Urist, Science, 150: 893 (1965). This observed activity was due to bone morphogenetic protein (BMP). Since the 1960s several investigators have attempted to identify and characterize this activity. Thus, a protein of 22 Kd, called osteogenin, was identified that possessed the activity. Sampath et al., Proc. Natl. Acad. Sci. USA, 84: 7109 (1987). Three proteins from demineralized ovine bone matrix were identified as having this activity. Wang et al., Proc. Natl. Acad. Sci., 85: 9484 (1988) and Wozney et al., Science, 242: 1528 (1988). These proteins were named BMP-1, BMP-2A, and BMP-3, the latter two of which belong to the extended TGF-.beta. family by limited sequence homology. These workers modified the assay for bone induction to show cartilage formation but did not show that the proteins ultimately stimulate formation of bone.
The TGF-.beta. group of molecules are each dimers containing two identical polypeptide chains linked by disulfide bonds. The molecular mass of these dimers is about 25 Kd. Biologically active TGF-.beta. has been defined as a molecule capable of inducing anchorage independent growth of target cell lines or rat fibroblasts in in vitro cell culture, when added together with EGF or TGF-.alpha. as a co-factor. TGF-.beta. is secreted by virtually all cell types in an inactive form. This latent form can be activated by proteolytic cleavage of mature TGF-.beta. from its precursor (at the Arg-Ala bond in position 278). A non-covalent complex is formed from the association of the mature TGF-.beta. with the precursor remainder or with a protein binding to TGF-.beta. or with alpha.sub.2 -macroglobulin. This complex is disrupted so as to activate the TGF-.beta. either by exposure to transient acidification or by the action of exogenous proteases such as plasmin or plasminogen activator.
There are at least five forms of TGF-.beta. currently identified, TGF-.beta.1, TGF-.beta.2, TGF-.beta.3, TGF-.beta.4, and TGF-.beta.5. Suitable methods are known for purifying this family of TGF-.beta.s from various species such as human, mouse, monkey, pig, bovine, chick, and frog, and from various body sources such as bone, platelets, or placenta, for producing it in recombinant cell culture, and for determining its activity. See, for example, Derynck et al., Nature, 316: 701-705 (1985); European Pat. Pub. Nos. 200,341 published Dec. 10, 1986, 169,016 published Jan. 22, 1986, 268,561 published May 25, 1988, and 267,463 published May 18, 1988; U.S. Pat. No. 4,774,322; Seyedin et al, J. Biol. Chem., 262: 1946-1949 (1987); Cheifetz et al, Cell, 48: 409-415 (1987); Jakowlew et al., Molecular Endocrin., 2: 747-755 (1988); Dijke et al., Proc. Natl. Acad. Sci. (U.S.A. ), 85: 4715-4719 (1988); Derynck et al., J. Biol. Chem., 261: 4377-4379 (1986); Sharples et al., DNA, 6: 239-244 (1987); Derynck et al., Nucl. Acids. Res., 15: 3188-3189 (1987); Derynck et al., Nucl. Acids, Res., 15: 3187 (1987); Derynck et al., EMBO J., 7: 3737-3743 (1988)); Seyedin et al., J. Biol. Chem., 261: 5693-5695 (1986); Madisen et al., DNA, 7: 1-8 (1988); and Hanks et al., Proc. Natl. Acad. Sci. (U.S.A. ), 85: 79- 82 (1988), the entire contents of these publications being expressly incorporated by reference.
TGF-.beta.3, TGF-.beta.4, and TGF-.beta.5, which are the most recently discovered forms of TGF-.beta., were identified by screening cDNA libraries. None of these three putative proteins has been isolated from natural sources, although Northern blots demonstrate expression of the corresponding mRNAs. TGF-.beta.4 and TGF-.beta.5 were cloned from a chicken chondrocyte cDNA library (Jakowlew et al., Molec. Endocrinol., 2: 1186-1195 [1988]) and from a frog oocyte cDNA library, respectively. The frog oocyte cDNA library can be screened using a probe derived from one or more sequences of another type of TGF-.beta.. TGF-.beta.4 mRNA is detectable in chick embryo chondrocytes, but is far less abundant than TGF-.beta.3 mRNA in developing embryos or in chick embryo fibroblasts. TGF-.beta.5 mRNA is expressed in frog embryos beyond the neurula state and in Xenopus tadpole (XTC) cells.
TGF-.beta. has been shown to have numerous regulatory actions on a wide variety of both normal and neoplastic cells. TGF-.beta. is multifunctional, as it can either stimulate or inhibit cell proliferation, differentiation, and other critical processes in cell function (M. Sporn, Science, 233: 532 [1986]). For a general review of TGF-.beta. and its actions, see Sporn et al., J. Cell Biol., 105: 1039-1045 (1987&gt;, Sporn and Roberts, Nature., 332: 217-219 (1988), and Sporn and Roberts, in Sporn and Roberts, ed., Handbook of Experimental Pharmacology: Peptide Growth Factors and Their Receptors I, Springer-Verlag, New York, pp. 3-15 (1990).
The multifunctional activity of TGF-.beta. is modulated by the influence of other growth factors present together with the TGF-.beta.. TGF-.beta. can function as either an inhibitor or an enhancer of anchorage-independent growth, depending on the particular set of growth factors, e.g., EGF or TGF-.alpha., operant in the cell together with TGF-.beta. (Roberts et al., Proc. Natl. Acad. Sci. U.S.A., 82: 119 [1985]). TGF-.beta. also can act in concert with EGF to cause proliferation and piling up of normal (but not rheumatoid) synovial cells (Brinkerhoff et al., Arthritis and Rheumatism, 26: 1370 [1983]).
Although TGF-.beta. has been purified from several tissues and cell types, as indicated above, it is especially abundant in bones (Hauschka et al., J. Biol. Chem., 261: 12665 [1986]) and platelets (Assoian et al., J. Biol. Chem., 258: 7155 [1983]). TGF-.beta. is postulated to be one of the local mediators of bone generation and resorption, because of its presence in large amounts in bone and cartilage, because cells with osteoblast and chondrocyte lineage increase replication after exposure to TGF-.beta., and because TGF-.beta. regulates differentiation of skeletal precursor cells. See Centrella et al., Fed. Proc. J., 2: 3066-3073 (1988).
Immunohistochemical studies have shown that TGF-.beta. is involved in the formation of the axial skeleton of the mouse embryo. TGF-.beta. is also present in other embryos in the cytoplasm of osteoblasts in centers of endochondral ossification and in areas of intramembranous ossification of flat bones, such as the calvarium. Heine et al., J. Cell. Biol., 105: 2861-2876 (1987). Following in situ hybridization of TGF-.beta.1 probes, localization of TGF-.beta. in both osteoclasts and osteoblasts has been described in development of human long bones and calvarial bones. Sandberg et al., Development, 102: 461-470 (1988); Sandberg et al., Devel. Biol., 130: 324-334 (1988). TGF-.beta. is found in adult bone matrix (Seyedin et al., Proc. Natl. Acad. Sci. USA, 82: 2267-2271 [1985], Seyedin et al., J. BIOL, Chem., 261: 5693-5695 [1986]) and appears at the time of endochondral ossification in an in vivo model of bone formation (Carrington et al., J. Cell. Biol., 107: 1969-1975 [1988]). Cultured fetal bovine bone osteoblasts as well as rat osteosarcoma cells have high mRNA levels for TGF-.beta. and secrete relatively high concentrations of TGF-.beta. (Robey et al., J. Cell. Biol., 105: 457-463 [1987]).
In certain in vitro models, TGF-.beta. was found to stimulate the synthesis of collagen, osteopontin, osteonectin, and alkaline phosphatase, and to stimulate replication in osteoblast-like cells. See Centrella et al., J. Biol. Chem., 262: 2869-2874 (1987); Noda et al., J. Biol. Chem., 263: 13916 (1988); Wrana et al., J. Cell. Biol., 106: 915 (1988); Noda et al., J. Cell. Physiol., 133: 426 (1987); Pfeilshifter et al., Endocrinology, 121: 212 (1987); Centrella et al., Endocrinology, 119: 2306 (1986); Roby et al., J. Cell. Biol., 105: 457 (1987). In other in vitro models, TGF-.beta. was found to inhibit proliferation and expression of alkaline phosphatase and osteocalcin. See, for example, Noda and Rodan, Biochem. Biophys. Res. Commun., 140: 56 (1986); Noda, Endocrinology, 124: 612 (1989).
Further, while Centrella et al., supra, showed increased collagen synthesis after treatment of osteoblasts from rat calvaria with TGF-.beta., Robey et al., supra, could not show increased synthesis of collagen in fetal bovine bone osteoblasts, postulating that the increased collagen production is secondary to the effects of TGF-.beta. on the proliferation of osteoblasts. In organ culture, TGF-.beta. was reported to stimulate bone resorption in neonatal mouse calvarias, but inhibit resorption in the fetal rat long bone system. See Tashjian et al., Proc. Natl. Acad. Sci. USA, 82: 4535 (1981); Pfeilshifter et al., J. Clin. Invest., 82: 680 (1988). TGF-.beta. activity was reported to be increased in cultures of fetal rat calvaria and in calvarial cells incubated with stimulators of bone resorption, such as parathyroid hormone, 1,25-dihydroxyvitamin D.sub.3, and IL-1 (Petkovich et al., J. Biol. Chem., 262: 13424-13428 [1987], Pfeilschifter and Mundy, Proc. Natl. Acad. Sci. USA, 84: 2024-2028 [1987]). Furthermore, it was reported that TGF-.beta. inhibits the formation of osteoclasts in bone marrow cultures. Chenu et al., Proc. Natl. Acad. Sci. USA, 85: 5683-5687 (1988). The showing that TGF-.beta. has effects on both osteoclasts and osteoblasts led Pfeilschifter and Mundy, supra, to propose that it is involved in the strict coupling of the processes of bone resorption and bone formation characteristic of the remodeling process in adult bone. It has also been postulated that the local acidic, proteolytic environment provided by the osteoclasts results in activation of matrix-associated latent TGF-.beta.. Oreffo et al., Calcified Tiss. Internatl., 42: Suppl:A15 (1988).
In view of the conflicting results reported for in vitro activities, it is not clear whether in vitro models can be used to predict the effects of TGF-.beta. on bone formation and resorption in vivo. See Roberts et al., Proc. Natl. Acad. Sci. USA, 82: 119 (1985).
Additional references reporting that TGF-.beta. promotes the proliferation of connective and soft tissue for wound healing applications include U.S. Pat. No. 4,810,691 issued Mar. 7, 1989, U.S. Pat. No. 4,774,228 issued Sep. 27, 1988, Ignotz et al., J. Biol. Chem., 261: 4337 (1986); Varga et al., Biochem. Biophys. Res. Comm., 138: 974 (1986); Roberts et al., Proc. Natl. Acad. Sci. USA, 78: 5339 (1981); Roberts et al., Fed. Proc., 42: 2621 (1983); U.S. Pat. No. 4,774,228 to Seyedin et al. TGF-.beta. stimulates the proliferation of epithelia (Matsui et al., Proc. Natl. Acad. Sci. USA, 83: 2438 [1986]; Shipley et al. Cancer Res., 46: 2068 [1986]); induces collagen secretion in human fibroblast cultures (Chua et al., J. Biol. Chem., 260: 5213-5216 [1983]); stimulates the release of prostaglandins and mobilization of calcium (Tashjian et al., Proc. Natl. Acad. Sci. USA, 82: 4535 [1985]); and inhibits endothelial regeneration (Heimark et al., Science, 233: 1078 [1986]).
In wound chambers implanted subcutaneously, TGF-.beta. increased DNA and collagen production. Sporn et al., Science, 219: 1329 (1983); Sprugel et al., Am. J. Pathol., 129: 601 (1987). Moreover, TGF-.beta. produced collagen fibrosis when injected subcutaneously (Roberts et al., Proc. Natl Acad. Sci. USA, 83: 4167-4171 [1986]) and promoted healing of skin incisions in rats (Mustoe et al., Science, 237: 1333 [1987]). Nevertheless, although TGF-.beta. induced chondrogenesis in muscle-derived cells in vitro (Seyedin et al., Proc. Natl. Acad. Sci. USA, 82: 2267 [1985]; Seyedin et al., J. Biol. Chem., 261: 5693 [1986]), it did not produce cartilage in vivo even when implanted with collagenous substrates, a system used for a long time as a bone induction model in animals (Sampath et al., Proc. Natl. Acad. Sci. USA, 84: 7109 [1987]; Howes et al., Calcif. Tissue Int., 42: 34 [1988]).
New studies have shown a time-dependent appearance of mRNA for TGF-.beta.1 at a fracture site in a rat and have localized the peptide immunohistochemically in the periosteum of the healing fracture; the same researchers reported that injections of TGF-.beta.1 into the periosteal area of the femur of young rats have caused significant formation of new cartilage. Bolander et al., New York Academy of Sciences, "Transforming Growth Factor-.beta.s: Chemistry, Biology and Therapeutics, May 18-20, 1989. It has been found that injections of TGF-.beta.1 into the parietal bone of young rats stimulated periosteal bone formation, resulting in a thickening of the calvarium. Noda et al., J. Cell. Biol., 107: 48 (1988).
TGF-.beta. was reported to stimulate local periosteal woven bone formation when injected daily onto the periostea of parietal bones of neonatal rats. Noda and Camilliere, Endocrinology, 124: 2991-2994 (1989). The fact that TGF-.beta. increases bone thickness when applied adjacent to periosteum in vivo is also reported in Joyce et al., J. Cell Biol., 110: 2195-2207 (1990); Marcelli et al., J. Bone Min. Rest., 5: 1087-1096 (1990); Mackie et al., Bone, 11: 295-300 (1990).
Certain researchers reported that TGF-.beta. does not induce bone formation unless it is administered concurrently with a cofactor, e.g., an osteoinductive factor purified from bovine demineralized bone. Bentz et al., supra, U.S. Pat. No. 4,843,063 issued Jun. 27, 1989 to Seyedin et al., and U.S. Pat. No. 4,774,322 issued Sep. 27, 1988.
The remodeling of bone with TGF-.beta. is also described by Centrella et al., J., Bone and Jt, Surg., 73A: 1418-1428 (1991). Multiple applications of TGF-.beta.1 to rat femur induced a profound stimulatory effect with increased deposition of bone at the site of injection. Joyce et al., J. Bone Min. Res., 4: 255-259 (1989). Additionally, a single local application of TGF-.beta.1 in a methylcellulose gel formulation to sites of cartilage damage accelerated the onset and increased the incidence of bone formation adjacent to the cartilage. Beck et al., J. Bone and Mineral Research, 6: 961-968 (1991). A single local application of this same formulation in the rabbit skull defect model increased the amount of bone formation in a dose-dependent manner when measured 28 days after injury. Beck et al., J. Bone Min. Res., 6: 1257-1265 (1991).
Phosphate biomaterials have been prepared and investigated in a number of forms. The most widely studied are biodegradable beta tricalcium phosphate (TCP) and hydroxyapatite. A detailed description of the variety of calcium phosphate compositions studied can be found in deGroot, Bioceramics of Calcium Phosphate, Boca Raton, Fla., CRC Press, 1983. TCP is used as an in vivo scaffold for bone repair. Perhaps the most consistent and desirable property of TCP as well as other calcium phosphate ceramics is biocompatibility. Also, calcium phosphate ceramics are able to bond directly to bone. Driskell, Proc. Ann. Conf. Biomed. Eng., 15: 199 (1973).
While TCP has low impact resistance, it has application as a bone graft substitute or extender to the extent that proper fixation can be included during the TCP resorption and bone repair processes. It has been demonstrated that TCP in granular form can be used as an autogenous bone extender in the repair of long-bone discontinuities in rabbits. Lemons et al., First World Biomat. Cong. (Baden, Austria), 1980, 4. 10.3 (Abstract). The surgically created defects filled with 50:50 TCP:autogenous bone healed in six weeks as compared with four to six weeks when autogenous bone alone was used. These results indicate that some applications of the granular TCP may be possible in humans where a degree of stress-bearing is a factor. Porous TCP has been applied in block form with some success in mandibular discontinuities in dogs. Tortorelli and Posey, J. Dent. Res., 60: Special Issue A: 601 (1981) (abstract).
The principal clinical application of TCP has been in dentistry. Powdered TCP has been used for initiating apical closure in teeth and for treating periapical defects. Biodegradables may play a role as carriers for bone-inductive agents or bone-cell chemotactic factors. Dipolar microspheres or packets of osteoprogenitor cells donated by an individual may be incorporated within a polymer or ceramic, and in conjunction with characterized bone inductive proteins can be expected to enhance bone repair and augmentation at any chosen skeletal site. Hollinger et al., Biodegradable Bone Repair Materials, 207: 290-305 (1986).
TGF-.beta. is typically formulated at an acidic pH at which it is active. Various methods for its formulation include adding 2-5% methylcellulose to form a gel (Beck et al., Growth Factors, 3: 267-275 [1990] reporting the effects on wound healing of TGF-.beta. in 3% methylcellulose), adding collagen to form an ointment or suspension (EP 105,014 published 4 Apr. 1984; EP 243,179 published 28 Oct. 1987; EP 213,776 published 11 Mar. 1987), or adding a cosmetically acceptable vehicle to the TGF-.beta. for a topical formulation (U.S. Pat. No. 5,037,643 issued 6 Aug. 1991).
Additionally, human topical applications containing growth factors such as TGF-.beta. are described in EP 261,599 published 30 Mar. 1988. A slow-release composition of a carbohydrate polymer such as a cellulose and a protein such as a growth factor is disclosed in EP 193,917 published 10 Sep. 1986. A formulation of a bioactive protein and a polysaccharide is described in GB Pat. No. 2,160,528 granted 9 Mar. 1988. An intranasally applicable powdery pharmaceutical composition containing an active polypeptide, a quaternary ammonium compound, and a lower alkyl ether of cellulose is described in EP 193,372 published 3 Sep. 1986. See also U.S. Pat. No. 4,609,640 issued 2 Sep. 1986 disclosing a therapeutic agent and a water-soluble chelating agent selected from polysaccharides, celluloses, starches, dextroses, polypeptides, and synthetic polymers able to chelate Ca and Mg; and JP 57/026625 published 12 Feb. 1982 disclosing a preparation of a protein and water-soluble polymer such as soluble cellulose. In addition, a method for entrapping enzymes in gel beads for use as a biocatalyst is described in U.S. Pat. No. 3,859,169. Also, a method for preparing polyvinyl alcohol gel intended as a transdermal vehicle for water-soluble synthetic drugs is disclosed in JP 62/205035 published 9 Sep. 1987.
A purified particulate bone mineral product for use in medicine impregnated with a gel-forming protein or polysaccharide such as gelatin is disclosed that may also carry one or more absorbed drugs such as transforming bone growth factor. WO 90/01955 published 8 Mar. 1990. Use of TGF-.beta. and a biocompatible controlled release polymer is described by Langer and Moses, J. Cell. Biochem., 45: 340-345 (1991). An osteoinductive pharmaceutical formulation comprising an anti-fibrinolytic agent such as epsilon amino acid caproic acid or other lysine analogue or serine protease inhibitor and a cartilage and/or bone inductive protein such as bone morphogenetic protein is disclosed in WO 91/19510 published 26 Dec. 1991. The formulation may additionally contain a growth factor such as TGF-.beta. and may be encased in a TCP matrix. Biologically active polypeptides based on TGF-.beta. sequences disclosed as useful Ln the treatment of wounds and bone fractures are described in WO 90/14359 published 29 Nov. 1990. In addition, TGF-.beta. has been disclosed as a treatment for gingivitis and periodontal disease in the form of implants, microspheres, an absorbable putty-like matrix, or a polymeric material having the drug impregnated thereon. WO 90/04974 published 17 May 1990. Compositions with activin, also optionally containing a TGF-.beta., a bone morphogenetic protein, or bone marrow, have been formulated with hydroxyapatite and TCP as a dental and orthopedic implant and for bone growth induction. WO 92/14481 published 3 Sep. 1992. Also, TGF-.beta. formulated for treatment of inflammatory disorders is described in EP 269,408 published 1 Jun. 1988. Additionally disclosed are cytokines such as TGF-.beta. bound to a solid support, which may include ceramics and polymeric materials as well as insoluble protein materials such as gelatin, collagen, or albumin. WO 90/09798 published 7 Sep. 1990.
Stable lyophilized formulations of polypeptide growth factors such as TGF-.beta. containing polymers to impart viscosity to a reconstituted solution or polysaccharides to stabilize against loss of biological activity are described in EP 308,238 published 22 Mar. 1989 and EP 267,015 published 11 May 1988, respectively. See also EP 335,554 published 4 Oct. 1989 on a cosmetic composition suitable for topical application to mammalian skin or hair that can contain collagen, a gelatin, and powders such as starch and aluminum silicates. Gels with polymeric material for providing viscosity that may contain a polypeptide growth factor such as TGF-.beta. are described in EP 312,208 published 19 Apr. 1989. Collagen-polymer conjugates in admixture with particulate matter such as TCP are described by WO 90/05755 published 31 May 1990. A controlled drug delivery system for placement in a periodontal pocket containing discrete microparticles comprising the drug (e.g., TGF-.beta.) and a polymer is described in EP 451,390 published 16 Oct. 1991. A bioactive compound associated with liposomes that may include TGF-.beta. is described in EP 393,707 published 24 Oct. 1990 and in Strassman et al., Clin. Exp. Immunol., 86: 532-536 (1991).
A sustained-release formulation containing an active ingredient such as TGF and collagen and a least one organic acidic compound is described in EP 326,151 published 2 Aug. 1989. TGF-.beta. in combination with a proteinaceous matrix that may comprise collagen and/or fibrinogen is described by WO 91/03491 published 21 Mar. 1991. A collagen sponge useful as an implant for a wound-healing matrix for TGF-.beta. and FGF is described in U.S. Pat. No. 4,950,483 issued 21 Aug. 1990. A therapeutic drug that contains a growth factor may be formulated in the form of powder, granules, etc., for example, with gelatin. JP 1-153647 published 15 Jun. 1989. Cicatrising compositions containing activated TGF-.beta. may be formulated with polysaccharides and humectants such as glycerol. FR 2,667,789 published 17 Apr. 1992.
It has also been known to mix an active medicament unstable to heat with a biodegradable protein carrier such as collagen, atelocollagen, or gelatin to form a carrier matrix having sustained-release properties. The resultant mixture is then dried, and the dried material is formed into an appropriate shape, as described in U.S. Pat. No. 4,774,091.
It would be desirable to provide a formulation for TGF-.beta. with the proper consistency suitable for molding to fill in bone gaps where needed.
Accordingly, it is an object of the present invention to provide a suitable formulation of exogenous TGF-.beta. to a local site on an animal where skeletal (bony) tissue is deficient so as to produce in every case mature, morphologically normal bone at the site of administration where it is needed.
It is another object to provide a bone-inducing composition that is clinically relevant for filling in smaller bone defects than is required for prosthetic devices.
It is further object to provide a TGF-.beta. formulation with enhanced consistency for improved application to the desired bone defect site.
These and other objects will become apparent to those skilled in the art.